Monday, June 23, 2014

PCR the molecular biologists best friend!

PCR (polymerase chain reaction) is an incredibly common method used in molecular biology. I'm not sure you can find a molecular biology lab that does not posses a thermocycler (the machine that runs PCR). PCR was invented in 1983 (which makes me older than PCR.........) and enhances small segments of DNA into millions and millions of copies. It is a powerful method because you can take a very small sample and end up with a great deal of DNA.

The first step to a PCR is to identify what segment of DNA you wish to amplify. For research, one is often amplifying a single gene of interest in the study. For my current work, I use primers that are designed to identify the GUS gene of my transformed plants. This lets me identify which plants have been successfully transformed as they will have the gene while non-transformed plants will not. Picking your primers is one of the most crucial steps in PCR. Primers are small segments of DNA that will pair with the genome DNA and direct the amplification of your gene. They must be designed so that they can only base pair in your gene of interest and not to other random segments of DNA, otherwise your PCR will give you meaningless information.

Once you have your primers and your DNA sample, then comes the master mix. The master mix will contain the primers, buffer, magnesium ions, dNTPS (extra nucleotides), and a polymerase. The one we use is hot start Taq. DNA Polymerase is the enzyme that builds new DNA. Taq stands for the species from which this polymerase comes from the bacteria Thermus aquaticus. T. aquaticus lives in hot springs and thus its enzymes do not degrade under increased temperature, like those in the cycling steps of PCR. 

Our thermocycler, an oldie but goodie
Once your DNA + master mix have been placed in the special PCR tubes, they will be transferred to the thermocycler where the real work happens. First, the tube will be raised up to 90+ degrees Celsius to activate Taq in the initiation step. Then, temperatures will bounce between 94, 50-65, and 72 degrees Celsius for a set number of cycles. The purpose of the temperature changes are to first denature all the DNA in the sample (94C), anneal the primers to the single stranded DNA (50-65C, the temperature in this step is determined by the primer sequence), and then DNA elongation by Taq polymerase using the primers as starting points (72C).
 With each cycle the amount of target DNA in the sample doubles until either the run ends, or all of the dNTPs are utilized. To calculate the number of target DNA produced you raise 2 to the cycle number power. My usual run is 22 cycles, 2^22 would equal 4,194,304 copies of the GUS gene. After cycling, the samples are held at 72C for a length of time determined by the length of the target gene to ensure that Taq finishes turning all the single stranded DNA into double stranded DNA. Then samples can be refrigerated/frozen until ready to use.

Loaded agarose gel
To check the quality of the PCR products, they are often run on an agarose gel. Agarose is a gelatinous matrix, similar to Jell-O, through which molecules move based on their size. Smaller particles move through the matrix faster than larger ones. Since you should know the size of your target DNA, you can easily check that your PCR product matches this expectation. It also gives a vague idea of how many copies, brighter bands = more copies.
Effect of cycle number on PCR products.
L to R 20, 22, 25, 28, 30 cycles.
Once the size is confirmed and everything looks good, then the PCR products can be used in several different processes. Personally, I'm just looking for yes/no so once I run the gel my PCR samples are no longer needed. But, back in my marine biology days I would take the PCR product and run it on a different gel to separate out small differences in my target DNA sequences to identify the algae type. They can also be used for cloning, sequencing, genetic testing, pathogen identification, and even crime scene investigations. The wide application of PCR products is what makes it a ubiquitous presence in molecular biology.

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